Rheumatoid arthritis (RA) is an auto-inflammatory disease that causes pain, swelling, stiffness, and loss of joint function. RA affects at least one percent (1%) of the U.S. population (about 2.5 million individuals). Currently, the cause of RA is unknown and while there is no cure, a certain level of control of RA can be achieved through the use of biologic drugs, physical exercise, joint protection techniques and self-management methods. Early diagnosis of RA and early, aggressive treatment can help prevent joint damage, deformity and loss of physical mobility. Confirmations of diagnoses are achieved through a combination of diagnostic tests, physical examination, x-ray evidence and imaging approaches.
Peptidyl arginine deiminases (PADs) have emerged as key participants in the pathogenesis of RA. PADs catalyze the post-translational deimination of peptidyl-arginine to citrulline in a reaction requiring calcium. Several observations highlight a central role for these enzymes including the following: (i) citrullinated proteins are hallmark targets of the autoantibody response in RA; (ii) PADs catalyze the post-translational deimination of peptidyl-arginine to citrulline; and (iii) PAD2 and PAD4 are expressed in synovial tissue from patients with RA in regions co-expressing citrullinated proteins, likely in neutrophils and monocytes.
PAD4 requires calcium for catalytic activity, and calcium activation of PAD4 displays positive cooperativity. Although in vitro citrullination assays typically use 5-10 mM calcium to achieve maximal PAD4 activation, it is not possible that such high calcium concentrations are present during PAD4 activation in vivo. Indeed, extracellular free calcium concentrations are estimated to be 0.49-0.98 mM in synovial fluid and 1.1-1.3 mM in plasma, and the maximum intracellular calcium concentration achieved by primary human cells even after stimulation with various stimuli does not exceed 100 μM. The discrepancy between the in vitro requirements and in vivo availability of calcium suggests that undiscovered binding-partners may modulate PAD4 calcium sensitivity in vivo during homeostasis and RA pathology.
Several studies have demonstrated that in addition to its role in protein citrullination, PAD4 is also a frequent antigenic target in RA. PAD4 autoantibodies are detectable prior to disease onset and are associated with more erosive RA that persists despite treatment with TNFα inhibitors. Although anti-PAD4 antibodies mark a subset of RA patients with severe disease, there is marked heterogeneity in disease severity observed amongst this group and a pathogenic role for these antibodies remains undefined.
Connective tissue disease associated with interstitial lung disease, or CT-ILD, is a lung condition that affects a small number of patients with a connective tissue disease. Examples of connective tissue diseases—also known as rheumatologic, collagen vascular or autoimmune diseases—include scleroderma, rheumatoid arthritis, Sjogren's syndrome, systemic lupus erythematosus, polymyositis, dermatomyositis and mixed connective tissue disease. Patients are often diagnosed with the connective tissue disease first and develop CT-ILD later, although in some cases, the opposite occurs. CT-ILD causes inflammation or scarring (fibrosis) of the lungs, or both. The exact cause of lung damage is unknown.
Antibodies targeting citrullinated proteins (ACPA) have been implicated in the pathogenesis of ILD in RA. Citrullinated proteins and the enzymes that catalyze citrullination, the peptidylarginine deiminases (PADs), are detected in RA-ILD lung tissue and even in the lungs of heavy smokers.
The present inventors have recently shown that peripheral blood neutrophils express PAD3 protein which is capable of citrullinating intracellular targets following cell activation with calcium and ionomycin. Due to the unexpected expression of PAD3 in neutrophils, the present inventors sought to investigate PAD3 as a potential autoantigen in RA. Such novel biomarkers would be useful in identifying patients with RA and who may have severe RA earlier in the disease progression. In addition, such biomarkers may be helpful in diagnosing RA patients who may be at risk for developing CT-ILD, or which may have undiagnosed CT-ILD and allow earlier intervening treatments.